1. Field of the Invention
The present invention is directed to a new class of polymeric compounds for binding to complementary DNA and RNA strands. In particular, the invention concerns compounds wherein naturally-occurring nucleobases or other nucleobase-binding moieties are covalently bound to an oligolactamtide, i.e., a backbone comprising repeating units that are phosphoric acid esters of .beta.-lactams. These compounds are useful for diagnostics, research reagents and therapeutics. The present invention is also directed to processes for synthesizing such compounds, and to intermediates used in such processes.
The ultimate mechanism of action for most conventional therapeutic agents, i.e. drugs, is by way of modulation of one or more targeted endogenous proteins, e.g., enzymes. Such agents, however, typically lack total specificity for their target proteins, and instead interact with other proteins as well. Thus, a relatively large dose of the agent must be used to effectively modulate a target protein; and the agent can cause undesired side effects as the result of interference in the action of the non-target proteins. Typical daily doses of such conventional agents are from 10.sup.-5 -10.sup.-1 millimoles per kilogram of body weight or 10.sup.-3 -10 millimoles for a 100 kilogram person. If this modulation instead could be effected by interaction with or inactivation at a point in the biological pathway at an earlier stage, a dramatic reduction in the necessary amount of the therapeutic agent necessary could likely be achieved, along with a corresponding reduction in side effects. Further reductions could be effected if such interaction could be rendered site-specific.
Proteins are produced in biological systems ultimately from genes that encode them. A gene performs its basic function by transcription of its encoded information to messenger RNA (mRNA) which, by interaction with the ribosomal complex in a synthetic process called translation, directs the assembly of the protein coded for by its sequence. Translation requires the presence of various co-factors and the amino acid building blocks for the protein, and their transfer RNAs (tRNA), all of which are present in normal cells.
In order for transcription to be initiated, there must be recognition of a specific promoter DNA sequence by the RNA-synthesizing enzyme, RNA polymerase. In many cases in prokaryotic cells, and probably in all cases in eukaryotic cells, this recognition is preceded by sequence-specific binding of a protein transcription factor to the promoter. Other proteins which bind to the promoter, but whose binding prohibits action of RNA polymerase, are known as repressors. Thus, gene activation typically is regulated positively by transcription factors and negatively by repressors. These genetic regulatory mechanisms and the ability to influence them have significant implications for diagnostics and therapeutics. Since a functioning gene continually produces mRNA, it is advantageous if gene transcription is modulated or blocked totally.
Oligonucleotides have been shown to interact with mRNA and other components associated with gene transcription. By virture of such interaction, synthetic preparations of naturally occurring oligonucleotides and synthetic oligonucleotide derivatives and analogs which do not occur in nature, have become valuable tools for research and important agents in diagnostic, therapeutic and other applications. Increasingly, there is a demand for such improved oligonucleotides, oligonucleotide analogs, as well as for methods for their preparation.
Oligonucleotides have been used in a number of areas of research. In genomic research oligonucleotides have been used as probes and primers. Oligonucleotides are also useful in devising diagnostics since they can specifically hybridize to nucleic acids of interest in the etiology of a given disease. Oligonucleotides are also being tested as therapeutic moieties in the treatment of disease states in animals and man. For example, workers in the field have now identified oligonucleotide therapeutic compositions that are capable of modulating expression of genes implicated in viral, fungal and metabolic diseases. It has now become routine to synthesize oligodeoxyribonucleotides and oligoribonucleotides having hundreds of base pairs (bp) by solid phase methods using commercially available, fully automatic synthesis machines. In short, oligonucleotides are important molecules having a large commercial impact in biotechnology and medicine. As a consequence, improved oligonucleotides and methods for the synthesis of improved oligonucleotides, which afford reduced cost and environmental impact, along with increased efficiency and convenience, are also in demand.
Unmodified oligonucleotides, i.e. natural phosphodiester linked oligonucleotides, are unpractical for many uses because they have short in vivo half-lives or they suffer from a limited ability to penetrate cell membranes. In order to improve half life as well as membrane penetration, a large number of variations in polynucleotide backbones have been undertaken. These variations include the use of methylphosphonates, monothiophosphates, dithiophosphates, phosphoramidates, phosphate esters, bridged phosphoramidates, bridged phosphorothioates, bridged methylenephosphonates, dephospho internucleotide analogs with siloxane bridges, carbonate bridges, carboxymethyl ester bridges, acetamide bridges, carbamate bridges, thioether, sulfoxy, sulfono bridges, various "plastic" DNAS, .alpha.-anomeric bridges, and borane derivatives. These analogues have various properties. Only a few have proved to have such a combination of properties that render them substitutes for natural oligonucleotides.
One of the most useful oligonucleotide analogues discovered to date is a class of compounds known as peptide nucleic acids. These compounds have been found to have enhanced hybridization to complimentary DNA and RNA strands as compared to most other known oligonucleotide analogues as well as nuclease and protease stability. Peptide nucleic acids have neutral, amide linked backbones. Retention of a phosphorous atom in oligomeric backbones is considered to be highly desirable for certain utilities since such backbones are capable of providing a charged species and for providing a potential site for interactions with peptides, as for instance with transcription factors.
The .beta.-lactam nucleic acid oligomers of the present invention are expected to exhibit superior properties as compared to prior reagents in that they mimic the higher affinity for complementary single stranded DNA (ssDNA) exhibited by peptide nucleic acids but, unlike peptide nucleic acids, the .beta.-lactam backbone is phosphorous linked resulting in a charged compound. The .beta.-lactam compounds are also expected to form triple helices where a first .beta.-lactam nucleic acid strand binds with RNA or ssDNA and a second .beta.-lactam nucleic acid strand binds with the resulting double helix or with the first .beta.-lactam nucleic acid strand. .beta.-lactam nucleic acids are generally water soluble to facilitate both diagnostic and research reagent use as well as cellular uptake. Moreover, .beta.-lactam nucleic acids contain the .beta.-lactam structure. Such structure is expected to make them biostable and resistant to enzymatic degradation, for example, by proteases.
With regard to the novel methods of preparation for the .beta.-lactam nucleic acids of the present invention, it is noted that methods have been employed heretofore for preparing oligonucleotides that utilize solid-phase synthesis wherein an oligonucleotide is prepared on a polymer or other solid support. Such solid-phase synthesis relies on sequential addition of nucleotides to one end of a growing oligonucleotide. Typically, a first nucleoside is attached to an appropriate support, e.g. long chain alkyl amine controlled pore glass (LCAA CPG), and nucleotide precursors are added stepwise to elongate the growing oligonucleotide. The nucleotide precursors are conventionally reacted with the growing oligonucleotide using the principles of a "fluidized bed" for mixing of the reagents, where solid supports composed of silica are used. While some of the techniques of such conventional processes are used in the novel methods of the present invention, nowhere is there a suggestion of the novel reactants and steps that characterize the present methods.